Male sterility associated with overexpression of the noncoding hsromega gene in cyst cells of testis of Drosophila melanogaster.

Of the several noncoding transcripts produced by the hsromega gene of Drosophila melanogaster, the nucleus-limited >10-kb hsromega-n transcript colocalizes with heterogeneous nuclear RNA binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles. Our earlier studies suggested that the noncoding hsromega-n transcripts dynamically regulate the distribution of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments. Here we show that a P transposon insertion in this gene's promoter (at -130 bp) in the hsromega05421; enhancer-trap line had no effect on viability or phenotype of males or females, but the insertion-homozygous males were sterile. Testes of hsromega05421; homozygous flies contained nonmotile sperms while their seminal vesicles were empty. RNA:RNA in situ hybridization showed that the somatic cyst cells in testes of the mutant male flies contained significantly higher amounts of hsromega-n transcripts, and unlike the characteristic fine omega speckles in other cell types they displayed large clusters of omega speckles as typically seen after heat shock. Two of the hnRNPs, viz. HRB87F and Hrb57A, which are expressed in cyst cells, also formed large clusters in these cells in parallel with the hsromega-n transcripts. A complete excision of the P transposon insertion restored male fertility as well as the fine-speckled pattern of omega speckles in the cyst cells. The in situ distribution patterns of these two hnRNPs and several other RNA-binding proteins (Hrp40, Hrb57A, S5, Sxl, SRp55 and Rb97D) were not affected by hsromega mutation in any of the meiotic stages in adult testes. The present studies, however, revealed an unexpected presence (in wild-type as well as mutant) of the functional form of Sxl in primary spermatocytes and an unusual distribution of HRB87F along the retracting spindle during anaphase telophase of the first meiotic division. It appears that the P transposon insertion in the promoter region causes a misregulated overexpression of hsromega in cyst cells, which in turn results in excessive sequestration of hnRNPs and formation of large clusters of omega speckles in these cell nuclei. The consequent limiting availability of hnRNPs is likely to trans-dominantly affect processing of other pre-mRNAs in cyst cells. We suggest that a compromise in the activity of cyst cells due to the aberrant hnRNP distribution is responsible for the failure of individualization of sperms in hsromega05421; mutant testes. These results further support a significant role of the noncoding hsromega-n transcripts in basic cellular activities, namely regulation of the availability of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments.

Developmental regulation and complex organization of the promoter of the non-coding hsr(omega) gene of Drosophila melanogaster.

The nucleus-limited large non-coding hsr(omega)-n RNA product of the 93D or the hsr(omega) gene of Drosophila melanogaster binds to a variety of RNA-binding proteins involved in nuclear RNA processing. We examined the developmental and heat shock induced expression of this gene by in situ hybridization of nonradioactively labelled riboprobe to cellular transcripts in intact embryos, larval and adult somatic tissues of wild type and an enhancer-trap line carrying the hsr(omega) 05241 allele due to insertion of a P-LacZ-rosy+ transposon at -130 bp position of the hsr(omega) promoter. We also examined LacZ expression in the enhancer-trap line and in two transgenic lines carrying different lengths of the hsr(omega) promoter upstream of the LacZ reporter. The hsr(omega) gene is expressed widely at all developmental stages; in later embryonic stages, its expression in the developing central nervous system was prominent. In spite of insertion of a big transposon in the promoter, expression of the hsr(omega) 05241 allele in the enhancer-trap line, as revealed by in situ hybridization to hsr(omega) transcripts in cells, was similar to that of the wild type allele in all the embryonic, larval and adult somatic tissues examined. Expression of the LacZ gene in this enhancer-trap line was similar to that of the hsr(omega) RNA in all diploid cell types in embryos and larvae but in the polytene cells, the LacZ gene did not express at all, neither during normal development nor after heat shock. Comparison of the expression patterns of hsr(omega) gene and those of the LacZ reporter gene under its various promoter regions in the enhancer-trap and transgenic lines revealed a complex pattern of regulation, which seems to be essential for its dynamically varying expression in diverse cell types.

Regulation of HSP70 in excitatory neurons: Possible implications for neuronal functioning.

Neurons maintain an intricate organization of cytoplasmic and membrane proteins for their integrity, quick communication across synapses and for other complex activities. Molecular chaperones such as members of the 70 kDa heat shock protein (HSP70) family may play very important roles in these functions. However, in spite of a recent report suggesting the presence of HSP70 related proteins in the synaptic vesicle docking complex at presynaptic sites and the known significant roles for HSP70 in excitotoxicity, there are remarkably few studies that have explored the potential role of HSP70 family proteins in physiological functions of neurons. Here we bring together direct and indirect evidences which suggest that several different pathways involved in long-term potentiation can influence the HSP70 levels at the synapse and hypothesize on possible physiological significance of this family of proteins in neuronal functions.

Heat shock but not benzamide and colchicine response elements are present within the — 844 bp upstream region of the hrsω gene of Drosophila melanogaster.

The selective inducibility of hsrω gene by heat shock and several chemical agents and its selective non-inducibility by heat shock under certain conditions led to suggestion that this locus is subject to multiple controls at the level of transcription. With a view to delimit these different control elements, transgenic lines horbouring hsrω 5’ promoter deletion variants tagged to the lacZ reporter gene were used. Three different assays, viz., staining for ß-galactosidase activity in different larval tissues using chromogenic X-gal substrate, [3H] uridine labelling of polytene nuclei and in situ DNA-DNA hybridization with a non-radioactive probe to polytene chrmosome spreads for checking the puffing status of the resident and the transgene in larval salivary glands, were applied to monitor the activiy of the reporter gene following different treatments. Our results showed that the – 844 bp to +107 bp sequence was sufficient for heat shock induction of the transgene in all tissues. An analysis of the base sequence of the hsrω promoter revealed the presence of three consensus heat shock elements at – 466, – 250 and at – 57 bp and of two GAGA factor binding sites at – 496 and at – 68bp within the – 844 bp region. Germline transformants carrying the – 346 bp to – 844 bp region of the hsrω promoter showed only a very weak heat shock inducibility of the reporter gene in agreement with the presence of only one of the three putative heat shock elements and one of the two GAGA factor binding sites in this region. Interestingly, neither of the transformed lines (carrying the – 844 bp to + 107 bp or the – 844 bp to –346 bp of the hsrω promoter region) showed any response of the transgene to benzamide or colchicine treatments. These results showed that while the heat shock response elements of the hsrω are included within the – 844 bp region the response elements for benzamide and colchicine treatments are outside this region.

hsp 83 mutation is a dominant enhancer of lethality associated with absence of the non-protein coding hsrω locus in Drosophila melanogaster.

The hrsω or the 93D heat shock locus of Drosophila melanogaster, which does not code for any protein, has an important role in development since nullosomy of this locus in transheterozygotes for two overlapping deficiencies, viz., Df(3R)eGp4 (eGp4) and Df(3R)GC14 (GC14), is known to cause a high (~ 80%) mortality with the small number of escapee nullosomic flies being sterile, weak and surviving for only a few days. We now show that a majority of the hsrω-nulosomics die as embryo and that the 20% escapee embryos develop slower compared to their sibs carrying either one or two copies of the hsrω locus but after hatching survive to pupal/imago stage. Most interestingly, we further show that when one hsp83 mutant allele (hsp83e4A) is introduced in eGp4/GC14 trans-heterozygotes, practically none of the hsrω-nullosomic embryos develop beyond the 1st instar larval stage. The specificity of this interaction between hsp83 and hsrω genes was further confirmed by examining the effect of the hsp83 mutant allele on other mutations in the 93D cytogenetic region. Therefore, we conclude that the hsp83 mutation acts as a dominant enhancer of the lethality associated with nullosomy for the hsrω gene. The observed genetic interaction between these two members of the heat shock gene family during normal embryonic development of Drosophila reveals novel aspects of their biological functions.

RNA polymerase II dependent genes that do not code for protein.

In recent years more and more examples of RNA polymerase II dependent non-coding transcripts have been described. Although these have frequently been ignored as "selfish DNA elements", it is becoming increasingly clear that many, if not all, of them have very important biological roles. Examples of such "genes" from Drosophila, mammals, other vertebrates, yeast etc. are considered. Although the specific mechanisms through which these non-coding transcripts function in the cell are not clear, comparisons reveal certain common themes, particularly the importance of secondary structures, rather than the primary base sequence of these transcripts. While some of these transcripts may function as ribozymes or as anti-sense regulators, most others may function more directly through their specific protein-binding properties. Since RNA is believed to be the first "living" molecule, it is very likely that some genes even today function only through this class of molecules. It is expected that instead of being ignored as examples of "selfish DNA", a more positive search for their functions will help unravel the significance of this novel class of genes.

Cell cycle and DNA content of mitotic cells in brain ganglia of Drosophila larvae.

The programmes of replication of hetero- and euchromatin regions, mitotic cell cycle and the DNA content in metaphases in brain ganglia from late third instar larvae of Drosophila melanogaster (wild type and a tumour bearing mutant, 1(2)gl, strain) and of Drosophila nasuta were examined by autoradiography of [3H]thymidine labelled (continuous or pulse) cells and by cytophotometry, respectively. Brain ganglia labelled continuously with [3H]thymidine for 24 h in vitro showed a significantly high proportion of cells with incorporation of radioactivity restricted to heterochromatin only. Pulse labelling of brain ganglia from larvae of Drosophila melanogaster and Drosophila nasuta followed by chase for different time intervals showed that (i) the frequency of labelled metaphases was more than 50% within 15 to 30 min of chase and remained higher than 50% in nearly all the chase samples till 24 h, (ii) euchromatin labelled metaphases appeared with a low frequency within 1 to 4 h chase period but the heterochromatin labelled metaphases continued to be more common in the later chase samples also, (iii) single chromatid labelled second cycle metaphases were seen within 1 to 4 h after the pulse, but their frequency did not increase in the later samples. Cytophotometry of feulgen-DNA and Hoechst 33258 stained metaphases in late third instar larval brain ganglia revealed a greater variation in the DNA content of individual metaphases, although the means were close to the expected 4 C content. It appears that in relation to the known asymmetric cell divisions of neuroblast and other neural cells, the mitotically active cells in brain ganglia comprise a heterogenous population with widely varying lengths of the different phases of cell cycle; some of them may not cycle regularly and may possibly have a discontinuous S-phase.

Restriction enzyme digestion of heterochromatin in Drosophila nasuta.

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types of Drosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while with TaqI, no such undigested band was seen. The AluI resistant 23 kb DNA hybridized in situ specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome of Drosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.

In situ study of chorion gene amplification in ovarian follicle cells of Drosophila nasuta.

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis in Drosophila melanogaster and Drosophila nasuta was examined by [3H] thymidine autoradiography and by in situ hybridization with chorion gene probes. When pulse labelled with [3H] thymidine, the follicle cells from stage 10-12 ovarian follicles of both Drosophila melanogaster and, Drosophila nasuta often showed intense labelling at only one or two sites per nucleus. In situ hybridization of chorion gene probes derived from Drosophila melanogaster with follicle cell nuclei of Drosophila melanogaster and Drosophila nasuta revealed these discrete [3H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.

Heat shock response in ovarian nurse cells of Anopheles stephensi.

Transcriptional and translational changes following temperature shock at 37, 39 or 41°C to ovarian cells of Anopheles stephensi were studied. Temperature shock at 39°C induced 6 puffs on polytene chromosomes in the nurse cells as revealed by [3H] uridine incorporation studies. Only the 2R-19B puff was induced at 37°C and was found to be a major temperature shock locus remaining most active at all the 3 temperatures tested. Other temperature shock loci were activated only at 39°C. There was progressive inhibi tion of general chromosomal transcription with the rise of temperature. Transcription was drastically inhibited at 41°C but all the temperature shock loci still remained relatively active. Examination of [35S]methionine labelled newly synthesized ovarian proteins using sodium dodecyl sulphate-polyacrylamide slab gels revealed that all the heat shock polypeptides except the HSP 70 were synthesized in ovarian cells even at control temperature (29°C). Temperature shock induced the synthesis of HSP 70 and elevated the levels of other heat shock polypeptides (82, 30, 29, 23 and 17 KD). Present results suggest that the threshold level for induction of a complete heat shock response in mosquitos is higher (39°C) than the other dipteran insects studied and that a 41°C treatment is not lethal as in the case of Drosophila, Chironomus etc. These features reflect the adaptations of mosquitos to tropical climate and their dietary habit of warm blood meal.